With a dramatic increase in the number of complete bacterial genomes in the public and private databases, there exists an outstanding opportunity for the identification of new antibacterial protein targets. Interestingly, the accumulation of genomic sequences has been punctuated by the realization that typically greater than one third of any genome cannot be annotated for function based on sequence similarity. Several of these "unknowns" have been shown to be genetically validated drug discovery targets. However, without a defined biochemical activity, unknowns that prove to be essential for growth and viability are intractable for the purposes of drug discovery. Biochemical assay of protein function is critical in the implementation of high-throughput screening to discover lead compounds and to the successful optimization of those leads to tight-binding inhibitors and ultimately to efficacious drugs. The proposed research aims to break new ground in the problem of defining general functional assays, and in understanding the biological activities of proteins of unknown function. The experimental basis for our proposed methodology is microcalorimetry. Calorimetry is an ideal method to screen for potential substrates because prior knowledge of the enzymatic reaction is not required. In this research proposal, we aim to demonstrate an innovative high-throughput calorimetric methodology for identifying substrates and ligands for enzymes of unknown function. Establishment of the technology proposed here enables a procedure for the identification of novel antibacterial drug candidates to combat drug resistant pathogens. PROPOSED COMMERCIAL APPLICATIONS: The invention disclosed here provides a method for identifying substrates and inhibitors for new enzyme targets identified through genomic sequencing. As applied to proteins of unknown function from bacterial genomes, the method of substrate profiling allows one to establish assays for anti-bacterial lead compound discovery in the pharmaceutical industry. The annual world wide market for antibiotic compounds is in excess of $23 billion. Single product sales can exceed $1 billion. The technology described here enables the discovery of novel antibiotics.